Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

Emerging infectious diseases have become a major global problem with public health and economic consequences. It is an urgent need to develop new anti-infective therapies. The natural diterpene carnosol exhibit a wide variety of interesting antibacterial and antiviral properties, and it is considered a theoretical inhibitor of COVID-19 Mpro. However, this compound is present in the family Lamiaceae in low quantities. To obtain carnosol in concentrations high enough to develop pharmacological studies, we evaluated the efficiency of a micropropagation protocol of Rosmarinus officinalis using a solid medium and a temporary immersion system (TIS), as well as the effect of 6-benzylaminopurine (6-BAP) and α-naphthaleneacetic acid (NAA) on the growth of shoots. Moreover, we developed and validated an analytical method to quantify carnosol using the H-point standard additions method in the high-performance liquid chromatography diode array detector (HPLC-DAD). After 30 days of culture, TIS produced the maximum number of shoots per explant (24.33 ± 1.15) on a liquid medium supplemented with 6-BAP at 5.0 mg L−1. Next, we also evaluated the effect of immersion time and frequency for TIS. After 72 days of culture, the best results were obtained with an immersion cycle of 1 min every 12 h, yielding 170.33 ± 29.40 shoots. The quantification of carnosol on the samples was performed at a flow rate of 1.2 mL min−1 using binary isocratic mobile phase system 60:40 (v/v) 10 mM formic acid (pH 3.0) (A) and acetonitrile (B) on a reverse-phase column. The content of carnosol in the in vitro cultures was around 8-fold higher than in the wild plant. The present study represents an efficient alternative method to obtain carnosol for its pre-clinical and clinical development.

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Improved Mass Multiplication of Rhodiola crenulata Shoots Using Temporary Immersion Bioreactor with Forced Ventilation

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-012-9542-x ◽

2012 ◽

Vol 166 (6) ◽

pp. 1480-1490 ◽

Cited By ~ 15

Author(s):

Yan Zhao ◽

Wei Sun ◽

Ying Wang ◽

Praveen K. Saxena ◽

Chun-Zhao Liu

Keyword(s):

Forced Ventilation ◽

Temporary Immersion Bioreactor ◽

Temporary Immersion ◽

Mass Multiplication ◽

Rhodiola Crenulata

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A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

Annals of Botany ◽

10.1093/aob/mcr033 ◽

2011 ◽

Vol 108 (8) ◽

pp. 1463-1475 ◽

Cited By ~ 56

Author(s):

D. A. Steinmacher ◽

M. P. Guerra ◽

K. Saare-Surminski ◽

R. Lieberei

Keyword(s):

Somatic Embryogenesis ◽

In Vitro Regeneration ◽

Secondary Somatic Embryogenesis ◽

Temporary Immersion System ◽

Temporary Immersion ◽

Peach Palm

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Daminozide Enhances Vigor and Steviol Glycoside Yield of Stevia (Stevia rebaudiana Bert.) Propagated in the Temporary Immersion Bioreactor

10.21203/rs.3.rs-1223930/v1 ◽

2022 ◽

Author(s):

Rizka Tamania Saptari ◽

Rizkita Rachmi Esyanti ◽

Riza Arief Putranto

Keyword(s):

Stevia Rebaudiana ◽

Feedback Regulation ◽

Optimum Concentration ◽

Temporary Immersion Bioreactor ◽

Temporary Immersion ◽

Steviol Glycoside ◽

Kaurenoic Acid ◽

Biosynthesis Gene ◽

Inhibitor Treatment ◽

Ga Biosynthesis

Abstract Stevia (Stevia rebaudiana Bertoni) contains sweet compound widely used as natural sweetener, steviol glycoside (SG). SG is a diterpenoid secondary metabolite synthesized from ent-kaurenoic acid, the same precursor of Gibberellin (GA). Therefore, in this study, a GA inhibitor, Daminozide (0, 10, 20 ppm) was used to block ent-kaurenoic acid conversion towards GA synthesis in attempt to increase SG content of stevia propagated in Temporary Immersion Bioreactor (TIB). Daminozide in 10 mg/L was observed to be the optimum concentration which increased biomass weight and SG content (stevioside and rebaudioside A) up to 40%. The treatment also increased transcripts accumulation of genes enrolled in SG biosynthesis, such as SrKA13H, SrUGT85C2, and SrUGT76G1, indicating SG pathway become more active due to the inhibition of GA pathway. Furthermore, the inhibition of GA was also indicated by the upregulated expression of GA biosynthesis gene (GA3ox) as the result of feedback regulation, and the downregulated expression of GA catabolism gene (GA2ox2) as the result of feed-forward regulation caused by inhibitor treatment.

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